The present invention relates generally to mixtures containing anthocyanic pigments and particularly to a process for stabilizing the anthocyanic pigments in such mixtures to sunlight-induced bleaching. The invention also relates to a novel photostable colorant composition produced by such process as well as to a novel food product containing such colorant composition.
Anthocyanic pigments, i.e. anthocyanins and anthocyanidins, have been found to account for the natural colors of many fruits, vegetables and flowers. Despite their widespread occurrence in nature, however, these pigments have not been widely used as colorants in foods because of both limited availability and, in many cases, poor stability and resultant color loss.
The instability of anthocyanins in food systems has been studied extensively [Markakis, Food Technology, 4, 437(1974); Hrazdina, Labersmith-Wiss. U. Technol., 7, 193 (1974)]. Anthocyanic pigments appear to be unstable in aqueous solutions at pH's above 4, at high temperature or in the presence of H.sub.2 O.sub.2 or ascorbic acid. In addition, although anthocyanic pigments are stable under normal laboratory lighting conditions, direct exposure to sunlight causes extensive degradation and resultant color loss. [Van Buren, et al., Am J. Enol. & Vitic., 19, 147 (1968)].
Obviously, a process for stabilizing anthocyanic pigments in food products would greatly expand the potential utility of such pigments especially in products which cannot be constantly maintained at low temperatures in the dark. Nevertheless, such a process has not been developed, perhpas due to a lack of understanding of the mechanism of photodegradation. Only the mechanism involved in decoloration by increasing pH appears to have been determined. [Brouillard and Dubois, J. Am. Chem. Soc., 99, 1359(1977); Brouillard and Delaportes, J. Am. Chem. Soc., 99, 8461(1977)].
It has long been known [Robinson and Robinson, Biochem. J., 25, 1687(1931)] that the various red to blue shades of flowers are due to anthocyanins either alone or in association with phenolic materials, called "co-pigments", which are present in the plants along with the anthocyanins. These co-pigments are known to cause a bathochromic shift in the .lambda..sub.max of the anthocyanin pigment and also an increase in the absorbance at .lambda..sub.max. Several phenolic compounds have been shown to give this "co-pigment effect" in model systems. [Asen, et al., Phytochem., 11, 1139(1972); Scheffeldt and Hrazdina, J. Food Sci., 43, 517(1978)]. This co-pigment effect appears to be at its greatest when the co-pigment is a flavonol, and the use of flavonol co-pigments, such as rutin and kaempferol-3-glucoside, has been suggested as a way to enhance the hue and intensity of anthocyanin colorants in foods [see Scheffeldt and Hrazdina, above]. The use of non-flavanoid compounds as co-pigments, however, does not appear to have been shown or suggested previously.
In order to determine the "co-pigment effect" of various compounds, both naturally occurring and synthetic, a 10 ppm solution of the representative anthocyanin, cyanidin rutinoside, in 0.01 M citric acid was combined with the compounds, and in the amounts, shown in Table I.
Both the .lambda..sub.max and the absorbance at .lambda..sub.max were measured for each mixture using a Beckman Model 25 UV-Visible spectrophometer and quartz cells. Absorbance readings were normalized according to the formula: Abs=[Abs w/copigment]/[Abs w.o. co-pigment].
__________________________________________________________________________ Effect of Various Co-pigments on the .lambda..sub.max (nm) and Absorbance* of a 10 ppm Solution of Cyanidin Rutinoside in 0.01 M Citric Acid. 0 20 50 100 200 500 1000 co-pigment ppm ppm ppm ppm ppm ppm ppm __________________________________________________________________________ 1. rutin 512 512 513 513 514 518 523 (1.0) (1.15) (1.16) (1.18) (1.22) (1.26) (1.32) 2. hydroxyethyl 512 513 514 516 518 523 528 rutin (1.0) (1.01) (1.02) (1.04) (1.05) (1.08) (1.10) 3. kaempferol-3- 512 513 514 516 521 528 533 glucoside (1.0) (1.01) (1.01) (1.04) (1.06) (1.08) (1.10) 4. quercetin 512 513 514 516 520 526 530 0-sulfates (1.0) (1.01) (1.03) (1.06) (1.10) (1.17) (1.23) 5. 4-methyl- umbelliferone 512 512 512 512 513 516 519 sulfate (1.0) (1.01) (1.02) (1.02) (1.05) (1.12) (1.20) 6. quercetin- 512 517 525 532 535 544 550 5'-sulfonate (1.0) (1.05) (1.07) (1.08) (1.08) (1.08) (1.08) 7. quercetin-5' 512 517 525 530 536 540 544 8- & 5', 6-di- (1.0) (1.07) (1.12) (1.16) (1.19) (1.19) (1.20) sulfonate 8. flavone mono- 512 514 517 519 522 528 535 sulfonate (1.0) (1.02) (1.05) (1.10) (1.16) (1.27) (1.33) 9. flavone di- 512 512 512 514 515 517 520 sulfonate (1.0) (0.99) (0.99) (0.99) (0.98) (0.96) (0.91) 10. 4'-methoxy-aurone mono- & di- 512 514 517 524 530 538 544 sulfonates (1.0) (1.01) (1.03) (1.06) (1.10) (1.10) (1.09) flavonol mono- & di- 512 513 514 516 518 522 527 sulfonates (1.0) (1.01) (1.02) (1.04) (1.08) (1.16) (1.23) morin di- 512 512 514 516 518 523 527 sulfonate (1.0) (1.04) (1.08) (1.14) (1.23) (1.35) (1.43) xanthone mono- 512 512 512 514 517 522 529 & di-sulfo- (1.0) (1.02) (1.02) (1.05) (1.11) (1.20) (1.26) nates N-methyl 512 513 515 519 522 527 531 acridone (1.0) (1.04) (1.09) (1.15) (1.21) (1.28) (1.28) mono- & di- sulfonates biochanin A 512 513 514 516 519 524 527 sulfonate (1.0) (1.02) (1.04) (1.07) (1.10) (1.14) (1.11) Anthraquinone 512 512 513 514 516 521 527 mono- & di- (1.0) (1.0) (1.02) (1.05) (1.10) (1.20) (1.31) sulfonates Apigenin mono- and di- 512 517 525 531 536 542 546 sulfonates (1.0) (1.06) (1.13) (1.18) (1.23) (1.26) (1.33) 4'-methoxy- flavone 512 515 518 522 528 536 543 sulfonate (1.0) (1.03) (1.06) (1.11) (1.17) (1.23) (1.26) __________________________________________________________________________ *absorbance expressed as [Abs. with copigment]/[Abs. without copigment].
In addition to the co-pigments shown in Table I, 1000 ppm concentrations of the following compounds were tested as in Table I and found to exert no spectral shift greater than 5 nm on 10 ppm solutions of cyanidin rutinoside: Chromone sulfonate, Catechin sulfonate, Dihydroquercetin sulfonate, Bis-(p-methoxy-benzoyl) methane sulfonate, 4-Hydroxycoumarin sulfonate, 7-Hydroxycoumarin sulfonate, Resorcinol sulfonate, p-Toluene sulfonic acid, Sulfosalicylic acid, Gallic acid, 4,4'-Dimethoxychalcone sulfonate, N-methylquinolone sulfonate.
Few, if any, naturally occurring flavonols are attractive for co-pigmentation studies because of their limited availability and/or limited water solubility. The most abundant flavonol, rutin (quercetin-3-rutinoside) (1), shows very poor solubility in water [130 ppm at room temperature-Krewson and Naghski, J. Am. Pharm. Assoc., 41, 582(1952)]. More soluble flavonols such as kaempferol-3-glucoside (3) are not available in sufficient quantities to meet commercial scale needs.
Conversion of rutin to a water soluble derivative has been achieved previously by reaction with 2-chloroethanol and NaOH to give mainly the tri-hydroxyethyl derivative, mixed with some di- and tetra-hydroxyethyl ethers. This mixture is generally known as hydroxyethylrutin (HER)(2). A sample of HER prepared by the Zyma procedure [British Patent No. 1,045,010] showed an effect very similar to that of rutin itself (Table I).
Quercetin, the aglycone of rutin, shows no co-pigmentation effect, presumably due to its extremely low solubility. Although hydroxyethylated quercetin has not been prepared, various sulfate esters have been. Quercetin-O-sulfates, i.e. a complex mixture of mono-, di-, tri-, and tetrasulfate esters (4) prepared from quercetin and sulfamic acid by the method of Yamaguchi [Nippon Kagaku Zasshi, 81, 1332(1960); Chem. Abs., 56, 445], show a distinct co-pigment effect (Table I).
As shown in Table I, the co-pigments which show the greatest co-pigment effect are the polyhydroxy flavonol sulfonates. Instead of esterifying the flavonol OH groups, sulfonation involves nuclear substitution of the flavone with the SO.sub.3 H group. Quercetin-5'-sulfonate (6) shows a co-pigmentation effect with cyanidin rutinoside at concentrations approximately 1/10 that at which rutin or HER show equivalent effects. This superiority for the quercetin-5'-sulfonate can best be seen by comparing the data for each co-pigment at the 100 ppm level. (Table I)
Quercetin-5'-sulfonate has been described many times previously, primarily as a reagent for spectrophotometric analysis of zirconium, hafnium, uranium and other elements. It has also been suggested as an ingredient in suntan lotion. For many years it was thought to be the 8-isomer, however, the correct 5' sulfonate structure was recently determined by NMR [Terpilowski, et al., Diss. Pharm. Pharmacol., 1970 (22), 389-93].
Although not quite as effective as quercetin-5'-sulfonate, quercetin-5',8-disulfonate (7) (which is prepared, along with some 5',6 disulfonate, as a by-product in the preparation of monosulfonate and isolated by prep HPLC) shows good co-pigment properties.
Each of these sulfonates, as well as others disclosed herein, are prepared by dissolving the parent compounds in a 50/50 mixture of conc. sulfuric acid and fuming sulfuric acid and allowing the mixture to stand at room temperature for approximately five minutes. The mixture is then poured into excess ice water and the solution neutralized with solid CaCO.sub.3. The resulting CaSO.sub.4 is removed by filtration and the filtrate passed through a strong cation exchange resin in the Na.sup.+ form to remove excess Ca.sup.++ ions. The eluent is then freeze-dried to yield the 40-60% pure sulfonate.
As shown by the data in Table I, several compounds in addition to flavanoids can function as co-pigments to enhance the color of foods containing anthocyanin pigments. It does not appear, however, that anyone has shown or suggested a solution to the more critical problem associated with anthocyaninc pigments, i.e., how to reduce or eliminate the tendency of these pigments to fade when exposed to sunlight.